Porphyromonas gingivalis persisters induce the immuno-inflammatory responses in macrophages by upregulating the forkhead box1 signaling pathway

Abstract

Objective To investigate the effects of Porphyromonas gingivalis (Pg) persisters (Ps) on immuno‑inflammatory responses in macrophages, and to explore the underlying mechanisms. Methods Pg cells were cultured to the stationary phase (72 h), and subsequently treated by high concentration of metronidazole at 100 mg/L, amoxicillin at 100 mg/L and the combination of them for different time period, named as metronidazole group, amoxicillin group and (metronidazole+amoxicillin) group. Pg cells without treatment were used as Blank control. The survival profile of PgPs cells was measured by colony‑forming unit assay. The living state of PgPs was observed by Live/Dead staining. Then, Pg and metronidazole‑treated PgPs (M‑PgPs) were used to treat macrophages, named as Pg group and M‑PgPs group. Transmission electron microscopy (TEM) was used to observe the bacteria in the macrophages. The expression levels of proinflammatory cytokines in macrophages were determined by real‑time fluorescence quantitative PCR and enzyme‑linked immunosorbent assay. The location of forkhead box transcription factor 1 (FOXO1) was detected by confocal immunofluorescence microscopy. After inhibiting or enhancing the FOXO1 expressions using inhibitors (Fi) or activators (Fa) respectively, the macrophages were treated with Pg and M‑PgPs, divided as Blank group, Pg group, M‑PgPs group, Fi group, (Fi+Pg) group, (Fi+ M‑PgPs) group, Fa group, (Fa+Pg) group and (Fa+M‑PgPs) group. Then, the expression pattens of proinflammatory cytokines were assessed. Results Remarkable number of lived PgPs was observed, both in planktonic culture and Pg biofilms either treated with metronidazole, amoxicillin or both, and those persisters could form new colonies. Pg and M‑PgPs were able to enter into the macrophages and the protein expression levels of interleukin (IL)‑1β, IL‑6, IL‑8 and tumor necrosis factor‑α (TNF‑α) [Pg group: (2 392±188), (162±29), (5 558±661), (789±155) μg/L; M‑PgPs group: (2 415±420), (155±3), (5 732±782), (821±176) μg/L] were significantly upregulated than those in Blank group [(485±140), (21±9), (2 332±87), (77±7) μg/L] (P<0.01). Moreover, Pg and M‑PgPs could facilitate the nuclear translocation and accumulation of FOXO1. In addition, the relative mRNA expression levels of FOXO1, B-cell lymphoma 6 and Krüppel-like factor 2 were upregulated when compared to Blank group (P<0.05). Furthermore, the protein expression levels of IL‑1β, IL‑6, IL‑8 and TNF‑α in Fi+Pg group [(1 081±168), (70±8), (1 976±544), (420±47) μg/L] were remarkably lower than Pg group [(4 411±137), (179±6), (5 161±929), (934±24) μg/L] (P<0.05). Similarly, the protein expression levels of IL‑1β, IL‑6, IL‑8 and TNF‑α in Fi+M‑PgPs group [(1 032±237), (74±10), (1 861±614), (405±32) μg/L] were remarkably lower than M‑PgPs group [(4 342±314), (164±17), (4 438±1 374), (957±25) μg/L] (P<0.05). On the contrary, the protein expression levels of IL‑1β, IL‑6, IL‑8 and TNF‑α in Fa+Pg group [(8 198±1 825), (431±28), (8 919±650), (2 186±301) μg/L] and Fa+M‑PgPs group [(8 159±2 627), (475±26), (8 995±653), (2 255±387) μg/L] were significantly higher than Pg group and M‑PgPs group, respectively (P<0.05). Conclusions PgPs are highly tolerant to metronidazole and amoxicillin. The M‑PgPs could enhance the immuno‑inflammatory responses in macrophages by upregulating the FOXO1 signaling pathway, while this effect exhibits no significant difference with Pg.

目的探索牙龈卟啉单胞菌（Pg）持留菌（Ps）对巨噬细胞免疫炎症反应的影响及其作用机制。

方法将Pg（ATCC 33277）悬浮培养和生物膜培养至对数末期（72 h）后，不使用药物处理以及使用高浓度甲硝唑（100 mg/L）和（或）阿莫西林（100 mg/L）处理不同时间，分别为空白对照组、甲硝唑组、阿莫西林组、甲硝唑+阿莫西林组，采用血平板计数法检测Ps生成数量，细菌活死染色检测Ps的生存状态；使用Pg和甲硝唑处理的PgPs（M-PgPs）刺激巨噬细胞，分别为空白对照组（不经任何处理）、Pg组、M-PgPs组；通过透射电镜观察细菌进入细胞内部的情况；使用实时荧光定量PCR（RT-qPCR）和酶联免疫吸附测定（ELISA）法检测巨噬细胞内炎症相关指标的表达情况；通过RT-qPCR检测巨噬细胞叉头盒转录因子1（FOXO1）信号通路的激活情况；利用共聚焦免疫荧光显微镜检测FOXO1在巨噬细胞内的分布情况。使用FOXO1抑制剂（Fi）和激活剂（Fa）处理巨噬细胞后，用Pg和M-PgPs刺激巨噬细胞，分别为空白对照组（不经任何处理）、Pg组、M-PgPs组、Fi组、Fi+Pg组、Fi+M-PgPs组、Fa组、Fa+Pg组和Fa+M-PgPs组，通过RT-qPCR和ELISA法检测巨噬细胞内炎症相关指标的表达情况。

结果悬浮培养和生物膜中的Pg经过高浓度的甲硝唑和（或）阿莫西林处理后，均无法被完全杀灭，且存活的Ps可重新生长形成菌落；Pg和M-PgPs均可进入巨噬细胞内部且巨噬细胞中的炎症因子白细胞介素（IL）-1β、IL-6、IL-8和肿瘤坏死因子α（TNF-α）的蛋白表达量［Pg组分别为（2 392±188）、（162±29）、（5 558±661）、（789±155）μg/L；M-PgPs组分别为（2 415±420）、（155±3）、（5 732±782）、（821± 176）μg/L］均显著高于空白对照组［分别为（485±140）、（21±9）、（2 332±87）、（77±7）μg/L］（均 P<0.01）。同时，Pg和M-PgPs均可促进巨噬细胞内FOXO1入核，巨噬细胞内FOXO1信号通路相关基因FOXO1、B细胞淋巴瘤6和Krüppel样转录因子2 mRNA的相对表达量均显著高于空白对照组（均 P<0.05）。另外，Fi+Pg组IL-1β、IL-6、IL-8和TNF-α的蛋白表达量［分别为（1 081±